Population Genetic Variation in Rare and Endangered Iliamna (Malvaceae) in Virginia
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BiologicalJournal OJ the Linnean Society (1996), 58: 357-369. With 4 figures Population genetic variation in rare and endangered IZiarnna (Malvaceae) in Virginia C. NEAL STEWART, JR., GARY ROSSON, BRENDA W. SHIRLEY AND DUNCAN M. PORTER Department of BioloD, Virginia Pohtechnic Institute and State Univerxip, Blacksburg, VA 24061-0406 USA. Reckued 28 March 1995, acceptedfor pubhatian 30 August 1995 Random amplified polymorphic DNA (RAPD) markers were used as input for an analysis of molecular variance (AMOVA), homogeneity of molecular variance analysis (HOMOVA), and cluster analysis to describe the population genetic structure of Iliamna cork, a federally endangered plant located only in Virginia, and I. rmota, a rare plant in Virginia, Indiana, and Illinois. The analysis was performed to help clarify the taxonomic relationship between the two closely related species. We analysed four clones in the only known population of I. cork, breeding stock derived from seeds originating from the population site, and three I. remota populations in Virginia. Eighty-five percent of screened primers revealed DNA polymorphisms in Iliamna. Ninety-nine informative markers were generated using seven primers. No significant statistical differences (at P = 0.05) in RAPD variation was found between species (24% of variance) using the AMOVA procedure. However, within specieslamong populations (31 % of the variance) and within populations (45%of the variance) there were significant differences (Pc 0.002). An unweighted paired group method using arithmetic averages (UPGMA) cluster analysis showed the federally endangered I. cork population to be genetically distinct from the apparently recently introduced (in Virginia: - 100ybp) I. remofa. The lack of significant differences from the AMOVA and the high number shared bands between I. cork and I. remofa suggest that I. cork may be more appropriately classified as a subspecies of I. remofa. Iliamna corei plants in the natural population were genetically similar to one another while the I. cork breeding stock plants and I. remota plants were genetically relatively diverse. 81996 The Linnean Society of London ADDITIONAL, KEY WORDS: -RAPD - random amplified polymorphic DNA ~ conservation genetics - plant taxonomy - mallow - AMOVA - analysis of molecular variance - endangered species. CONTENTS Introduction .......................... 358 Material and methods ....... ................ 360 Sampling strategy ....... ................ 360 RAF'D profiling and statistical analyses ................ 360 Results ............................ 36 1 RAPD profiling ....... ................ 36 1 Statistical analyses ...... ................ 364 Discussion ........................... 365 Acknowledgements ........ ................ 367 References ........... ................ 368 Correspondence to C. Neal StewartJr., present address: Department of Biology, Univenity of North Carolina, Greensboro, NC 274 12-5001, U.S.A. 357 0024-4066/96/070357+ 13 $18.00/0 01 996 The Linnean Society of London 358 C.N. STEWART ETA. INTRODUCTION Zliamna is a North American genus of the Malvaceae containing seven or eight species. Most of the species are indigenous west of the Mississippi River. However, two species are found exclusively in the east: I. corei and I. rmota. They have showy, insect-pollinated, perfect flowers, and are self-incompatible (T. Wieboldt and J. Randall, unpublished data). Iliumna corei (Shed) SherfT, the Peters Mountain mallow, is a federally endangered plant species that exists naturally in a single population, on Peters Mountain, Giles County, Virginia, USA) (Fig. 1). This population consists of four clumps, each comprising a clone (Stewart & Porter, 1995). The Peters Mountain site is a sandstone cliff (1000m elevation) above the New River. Seeds were unearthed from the site and germinated. The resulting plants were used to establish a breeding population on the campus of Virginia Polytechnic Institute and State University, Blacksburg, Virginia. Iliumna rmota Greene, the Kankakee mallow, is a rare species that was first collected on Altorf Island in the Kankakee River, Illinois (Strausbaugh & Core, 1932). The species apparently has spread eastward along railroad lines to Virginia (Porter & Wieboldt, 1991). In contrast with the mountain- dwelling Z; corei, the Virginia I. remota populations are located near the James River, not unlike the I. remota habitat in Illinois. Since the discovery of I. cork (Strausbaugh & Core, 1932), the proper taxonomic placement of the taxon has been in question. Strausbaugh & Core (1932) placed I. Figure 1. The sites sampled for mallow DNA in Vuginii, USA. See Table 3 for population abbreviations. RAPD VARIATION IN IWA 359 cord in the same species as I. remota (Phymosia remota (Greene) Britton) on the basis of similar morphological features. Shed€ (1 946) revised the classification such that the Kankakee mallow was recognized as I. remota var. ppica and the Peters Mountain mallow was renamed I. remota var. cord Sherff. The basis for this split was a difference in leaf morphology (the Kankakee mallow leaf typically has a broadly triangular terminal lobe subtended by obtuse sinuses, while the Peters Mountain mallow leaf has an oblong terminal lobe with sharp sinuses), leaf size (Kankakee mallow is larger than Peters Mountain mallow), and plant height (Kankakee mallow, l.CLl.7m; Peters Mountain mallow, 0.6-0.9 m). Three years later SherfY(1949)further split the taxa into two species, I. remota Greene and I. cord (She4 SherK, on the additional characters of differing corolla colors (I. cord has a deeper hue), and floral odor (I. remota is scented whereas I. cord is not). Therefore, the division of I. remota and I. cord was based upon very few different morphological characters observed on plants growing in ecologically diverse natural habitats. It is conceivable that the characters used by SherfF in separation of the species are plastic. Plastic traits are defined as those that vary in response to spatial (edaphic and geographic), and temporal heterogeneity, and are known to confound taxonomic classifications (Schlichting, 1986). For example, we have noticed that plants in the breeding population are taller than those on Peters Mountain. Likewise, Bounds (1992) reported that there are statistically significant in situ morphological differences between the population of I. corei and I. remota populations in Virginia. In addition, Strausbaugh & Core (1932) reported the height of I. Cora' on the mountain site approached 2m, although in recent years, the stems have been shorter. Common garden studies would be appropriate to control for differences among Iliamna sites. However, to our knowledge, no data stemming from common garden comparisons between I. cord and I. remota have ever been published, although Sherf€ (1949) and Scott (1973) reported growing them together in such a situation. An alternative to phenotype analysis to delineate taxa is to perform genetic analysis, which is, by nature, not affected by environment. However, there has been limited work using genetic markers in Iliamna. Bounds (1988) reported apparent polymorphisms in five isozymes of I. cord and I. remota with interclonal variation found in I. cord. In a preliminary random amplified polymorphic DNA (RAPD) study, two primers were used to distinguish clones (Stewart & Porter, 1995). No formal population-level analysis has been published to date. Methods have recently been developed to utilize RAPD profiling (Welsh & McClelland, 1990; Williams et al., 1990) in formal population genetic analyses (Lynch & Milligan, 1994; Stewart & Excoffier, 1996). Although numerous DNA polymorphisms may easily by revealed using RAPDs, the markers are primarily dominant, i.e. homozygotes are indistinguishable from heterozygotes (Tinker, Fortin & Mather, 1993; Williams et al., 1993). Stewart & Excoffier (1996) have modified the analysis of molecular variance (AMOVA) technique (Excoffier, Smouse & Quattro, 1992), adapting it for use with dominant markers. In short, they used estimated amounts of autogamy (selfing frequency from 0 to 1.0) to estimate the average genotype frequencies from the phenotypic (RAPD) data. These estimates were then used in the AMOVA. The AMOVA partitions variance to hierarchical levels, e.g. among and within populations, and tests for significance at these predefined levels, similar to an analysis of variance (ANOVA). The objectives of our study were threefold: (1) identlfjr population-level genetic markers in I. cord and I. remota; (2) provide baseline data on the genetic variability of 360 C. N. STEWART ETA. I. corei and I. remota and (3) cia* the taxonomic relationship between I. remotu and I. corei. Our approach was to use RAPD markers in a population genetic analysis. This represents the first use of the AMOVA for RAPDs to analyse an obligately outcrossing plant species, although Stewart & Excoffier (1996) used it for Vuccinium macrocarpon (Ericaceae), a selfing species, and Huff, Peakall & Smouse (1993) used an earlier version of AMOVA, which had not been modified for RAPD data, to analyze obligate outcrossing Buchloe dac&loides (Poaceae). Our approach was to assume that each surveyed population of Iliamna (not including the one I. riuularir sample we included for comparative purposes) were members of a panmictic metapopulation. The AMOVA, a permutational statistics package, tests for population substructuring and will indicate whether significant molecular differences exist within and among local breeding populations.